Widespread antibiotic use has accelerated S. aureus resistance to almost all marketed antibiotic classes including beta-lactams, fluoroquinolones, macrolides, aminoglycosides, tetracyclines, as well as the newer linezolid and daptomycin. Methicillin-resistant S. aureus (MRSA) is an increasing public health threat, with deaths from MRSA infections already comparable to HIV/AIDS in 2005, and increasing since then. Thus, there is a clear imperative for developing new therapeutic agents. In recent work, we have developed antimicrobial compounds that show low micromolar antibacterial activity against both wild type and methicillin-resistant S. aureus through dual mechanism activity - initially inducing rapid depolarization of bacterial membranes, followed by cell penetration and target-based bacterial killing through inhibition of the enzyme enoyl reductase, or FabI. Our inhibitors show mid- to low-nanomolar inhibition of FabI, with promising metabolic stability and cytotoxicity for our initial compounds. Based on extensive preliminary studies, including crystal structures of multiple inhibitors co-crystallized with FabI, mechanistic characterization of a unique mode of inhibition, and characterization of inhibitor metabolic stabilities, we now propose to develop improved antimicrobial agents through an iterative process of determining detailed structure-based molecular design, synthetic medicinal chemistry, initial preclinical toxicology, and pharmacokinetic characterization, with specific proposed milestones and go/no-go criteria. The goal of this Phase I STTR application is to develop proof of concept for antibacterial lead compounds against drug resistant S. aureus that are safe and efficacious.